阅读说明：本技术 一种利用玉米芯同步糖化发酵生产d-1,2,4-丁三醇的方法 (Method for producing D-1,2, 4-butanetriol by using corncobs through synchronous saccharification and fermentation ) 是由 陈可泉 李孟阳 王昕� 于 2021-02-24 设计创作，主要内容包括：本发明提供了一种利用玉米芯同步糖化发酵生产D-1,2,4-丁三醇的方法,该方法通过构建克隆表达2-酮酸脱羧酶和D-木糖脱氢酶,木糖酸脱水酶和醇脱氢酶的基因,并将构建好的基因转入宿主菌的细胞内得重组大肠杆菌,培养重组大肠杆菌并接种至含有经预处理过的玉米芯的培养基中发酵生产D-1,2,4-丁三醇。本发明以玉米芯为原料,同步糖化发酵生产丁三醇,D-1,2,4-丁三醇产量为6.44g/L。利用玉米芯同步糖化发酵生产1,2,4-丁三醇,操作简便,产量较高,适合产业化生产。(The invention provides a method for producing D-1,2, 4-butanetriol by simultaneous saccharification and fermentation of corncobs, which comprises the steps of constructing and cloning genes for expressing 2-keto acid decarboxylase, D-xylose dehydrogenase, xylonic acid dehydratase and alcohol dehydrogenase, transferring the constructed genes into cells of host bacteria to obtain recombinant escherichia coli, culturing the recombinant escherichia coli, and inoculating the recombinant escherichia coli into a culture medium containing pretreated corncobs for fermentation production of the D-1,2, 4-butanetriol. The invention takes corncobs as raw materials, and the yield of D-1,2, 4-butanetriol is 6.44g/L by synchronous saccharification and fermentation. The method for producing the 1,2, 4-butanetriol by using the corncobs through synchronous saccharification and fermentation is simple and convenient to operate, high in yield and suitable for industrial production.)

1. A method for producing D-1,2, 4-butanetriol by utilizing corncobs to carry out simultaneous saccharification and fermentation is characterized in that genes for cloning and expressing 2-keto acid decarboxylase, D-xylose dehydrogenase, xylonate dehydratase and alcohol dehydrogenase are constructed, the constructed genes are transferred into cells of host bacteria to obtain recombinant escherichia coli, the recombinant escherichia coli is cultured and inoculated into a culture medium containing pretreated corncobs to carry out fermentation production of the D-1,2, 4-butanetriol.

2. The method for producing D-1,2, 4-butanetriol by using the simultaneous saccharification and fermentation of the corncobs as the claim 1, which is characterized by comprising the following specific steps of:

step 1, constructing, cloning and expressing 2-keto acid decarboxylase, D-xylose dehydrogenase, xylonate dehydratase and alcohol dehydrogenase to obtain recombinant escherichia coli; step 2, pretreating the corncobs, treating the suspension hydrolysate obtained by mixing the sodium hydroxide solution with the corncobs at 121 ℃, filtering to obtain corncob residues, and washing the residues to be neutral by using pure water;

step 3, preparation of fermentation medium

Preparing LB culture medium of 5g/L yeast powder, 10g/L peptone and 5g/L sodium chloride, packaging in a 500mL conical flask, filling 50mL liquid, adding 2g pretreated corncob residues into the LB culture medium to prepare a fermentation culture medium, and sterilizing for later use; and 4, inoculating the recombinant escherichia coli into a fermentation culture medium, adding hemicellulase, and then adding IPTG (isopropyl-beta-thiogalactoside) to induce fermentation to obtain a product D-1,2, 4-butanetriol.

3. The method for producing D-1,2, 4-butanetriol by simultaneous saccharification and fermentation of corncobs as claimed in claim 2, wherein the host bacterium in step 1 is Escherichia coli Trans 1T 1.

4. The method for producing D-1,2, 4-butanetriol by using the simultaneous saccharification and fermentation of the corncobs as the claimed in claim 2, wherein the concentration of the sodium hydroxide in the step 2 is 1mol/L, and the mass-to-volume ratio of the corncobs to the sodium hydroxide solution is 1: 10.

5. The method for producing D-1,2, 4-butanetriol by using the simultaneous saccharification and fermentation of the corncobs as the claimed in claim 2, wherein the inoculation amount of the recombinant escherichia coli in the step 4 is 4%, the final concentration of IPTG is 1mmol/L, the initial culture temperature is 37 ℃, and the culture is carried out at 33 ℃ after the inducer is added until the fermentation is finished.

6. The method for producing D-1,2, 4-butanetriol by using the simultaneous saccharification and fermentation of the corncobs as claimed in claim 2, wherein the amount of the hemicellulase added in the step 4 is 400 IU.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for producing D-1,2, 4-butanetriol by simultaneous saccharification and fermentation of corncobs.

Background

D-1,2, 4-butanetriol is a colorless, odorless, transparent, viscous four-carbon polyol; has high solubility in water and alcohol, and is hygroscopic. In military, D-1,2, 4-butanetriol can be used for synthesizing butanetriol trinitrate (BTTN) serving as a rocket propellant, in the aspect of medicine, D-1,2, 4-butanetriol can be used as a slow release agent to prepare an intermediate of an antiviral compound, and D-1,2, 4-butanetriol can also be used as a cross-linking agent of a high polymer material to increase the strength and hardness of the material.

At present, the production of D-1,2, 4-butanetriol is mainly focused on chemical synthesis, i.e. using NaBH₄Reducing malic acid and derivatives thereof. However, the chemical synthesis of D-1,2, 4-butanetriol requires H at 2900-₂Under the pressure and the temperature of 60-160 ℃, the reaction conditions are very harsh, the operation has safety threat, and the environmental pollution is serious. Therefore, the development of a method for commercially producing D-1,2, 4-butanetriol is continued.

The biological synthesis of D-1,2, 4-butanetriol has attracted attention at present because of its advantages of safety, reliability, and easily available source. The D-1,2, 4-butanetriol synthesized by the biological method is mainly synthesized by taking xylose as a substrate through four-step enzyme reaction of D-xylose dehydrogenase, xylonic acid dehydratase, 2-keto acid decarboxylase and alcohol dehydrogenase. At present, two methods are mainly involved in synthesizing D-1,2, 4-butanetriol by a biological method: one is a whole-cell catalysis method, in which a large number of microorganisms expressing the four enzymes are cultured, cells are collected after the culture is finished, and xylose is converted into D-1,2, 4-butanetriol by using the collected cells; the method has the advantage of higher conversion rate, but the steps in the process are complicated, so that the method is not beneficial to large-scale production; the other method is to add substrate xylose during the process of culturing the microorganism expressing the four enzymes to directly produce BT. The BT produced by directly fermenting microorganisms has low cost, is simple and convenient to operate in the process and is beneficial to large-scale production.

Corncob is a common agricultural waste, and the main components of the corncob comprise cellulose, hemicellulose, lignin and the like, wherein the hemicellulose is xylan type and can be converted into xylose after being treated by hemicellulase, and the application value and the economic potential of the corncob are very huge. The corn cob is used as a raw material and is converted into a target product through microbial fermentation, so that two ways can be provided: and carrying out step-by-step saccharification fermentation and synchronous saccharification fermentation. The corn cob is saccharified firstly by the step-by-step saccharification and fermentation by using hydrolysate containing monosaccharide. CN106148429B discloses a method for producing D-1,2, 4-butanetriol by biotransformation of cellulose hydrolysate. The method comprises the steps of constructing and cloning genes for expressing 2-keto acid decarboxylase and D-xylose dehydrogenase, xylonate dehydratase and alcohol dehydrogenase, transferring the constructed genes into cells of host bacteria with xylose isomerase knocked out to obtain genetically engineered bacteria, culturing the genetically engineered bacteria, inoculating the genetically engineered bacteria into cellulose hydrolysate, and fermenting to produce the D-1,2, 4-butanetriol. The method adopts dilute acid to pretreat corncobs to obtain hydrolysate containing xylose, furfural and other fermentation inhibitors which are unfavorable for the growth of strains can be generated in the dilute acid pretreatment process, the pH value of the hydrolysate is very low, and the hydrolysate can be applied to the fermentation process only by the steps of neutralizing sulfuric acid with calcium hydroxide, detoxifying with activated carbon and the like in advance. The steps are more, which is not beneficial to large-batch fermentation. The simultaneous saccharification and fermentation couples the enzymolysis process with the fermentation process, and the corncob enzymolysis and the strain fermentation are carried out simultaneously to form the simultaneous saccharification and fermentation process, so the method has the advantages of simple and convenient operation, low production cost and larger commercial production space.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a method for producing D-1,2, 4-butanetriol by using corncobs through synchronous saccharification and fermentation, which reduces the production cost and improves the yield of the 1,2, 4-butanetriol.

In order to solve the problems of the prior art, the invention adopts the technical scheme that:

a method for producing D-1,2, 4-butanetriol by simultaneous saccharification and fermentation of corncobs comprises the steps of constructing and cloning genes for expressing 2-keto acid decarboxylase, D-xylose dehydrogenase, xylonate dehydratase and alcohol dehydrogenase, transferring the constructed genes into cells of host bacteria to obtain recombinant escherichia coli, culturing the recombinant escherichia coli, inoculating the recombinant escherichia coli into a culture medium containing pretreated corncobs, and fermenting to produce the D-1,2, 4-butanetriol.

As an improvement, the method for producing the D-1,2, 4-butanetriol by using the corncobs for simultaneous saccharification and fermentation comprises the following specific steps:

step 1, constructing clone expression 2-keto acid decarboxylase (mdLC), D-xylose dehydrogenase (xylB), xylonic acid dehydratase (yjhG) and alcohol dehydrogenase (adhP), and transferring the constructed genes into cells of host bacteria to obtain recombinant escherichia coli;

step 2, pretreatment of corncobs

Treating the suspension hydrolysate obtained by mixing the sodium hydroxide solution and the corncobs at 121 ℃, filtering to obtain corncob residues, and washing the residues to be neutral by using pure water;

step 3, preparation of fermentation medium

Preparing LB culture medium of 5g/L yeast powder, 10g/L peptone and 5g/L sodium chloride, packaging in a 500mL conical flask, filling 50mL liquid, adding 2g pretreated corncob residues into the LB culture medium to prepare a fermentation culture medium, and sterilizing for later use;

and 4, inoculating the recombinant escherichia coli into a fermentation culture medium, adding hemicellulase, and then adding IPTG (isopropyl-beta-thiogalactoside) to induce fermentation to obtain a product D-1,2, 4-butanetriol.

Preferably, the host bacterium in step 1 is Escherichia coli Trans 1T 1.

Preferably, the concentration of the sodium hydroxide in the step 2 is 1mol/L, and the mass-to-volume ratio of the corncobs to the sodium hydroxide solution is 1: 10.

Preferably, the inoculation amount of the recombinant Escherichia coli in the step 4 is 4%, the final concentration of IPTG is 1mmol/L, the initial culture temperature is 37 ℃, and after adding the inducer, the recombinant Escherichia coli is cultured at 33 ℃ until the fermentation is finished.

Preferably, the amount of hemicellulase added in step 4 is 400 IU.

Has the advantages that:

in the prior art, xylose is usually used as a substrate for producing D-1,2, 4-butanetriol, the price is higher, and the cost is usually higher when the xylose is applied to large-scale production. Corncobs contain a large amount of hemicellulose, and a large amount of xylose can be released through enzymolysis, but the corncobs are compact in structure and low in enzymolysis efficiency. Compared with dilute acid pretreatment for directly releasing a large amount of xylose, the alkaline pretreatment mainly has the function of breaking the compact structure of the corncobs, so that the hemicellulose is easily hydrolyzed by hemicellulase, fermentation inhibitors cannot be generated in the enzymolysis process, and the adverse effect on the fermentation process is less. The enzymolysis process is coupled with the fermentation process, the corncob enzymolysis and the strain fermentation are synchronously carried out, the synchronous saccharification and fermentation process is formed, the operation is simple and convenient, the production cost is low, and the large commercial production space is provided.

The invention takes the corncob as the raw material, alkali pretreats the corncob, so that the enzymolysis efficiency is improved, and the D-1,2, 4-butanetriol is produced by using the pretreated residue synchronous saccharification and fermentation method, so that the yield of the D-1,2, 4-butanetriol is improved, the cost is greatly saved, the operation is simple and convenient, and the potential of large-scale production is realized.

Drawings

FIG. 1 shows the production of D-1,2, 4-butanetriol by fermentation of recombinant Escherichia coli using xylose as a substrate;

FIG. 2 shows xylose yields after enzymolysis of pre-treated corncobs and post-treated residues for 24 hours;

FIG. 3 shows the production of D-1,2, 4-butanetriol by simultaneous saccharification and fermentation of recombinant Escherichia coli using corncob as a substrate.

Detailed description of the preferred embodiments

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.

In the following examples, the enzymes and plasmids used were purchased unless otherwise specified.

The 2-keto acid decarboxylase (mdlc), GenBank: AY 143338.1; d-xylose dehydrogenase (xylB), Gene ID: 7329904; xylonic acid dehydratase (yjhG), Gene ID: 946829; alcohol dehydrogenase (adhP), Gene ID: 00946036.

In addition, the term "simultaneously" as used herein means that saccharification of cellulose and fermentation of monosaccharides are performed simultaneously.

Example 1 construction of YjhG Gene-containing Strain

(1) Introducing enzyme cutting sites (NcoI and Hind III) by using primers at the 5 'end and the 3' end of the YjhG gene, carrying out double enzyme cutting on the YjhG gene and the pCWJ plasmid, and then connecting the YjhG gene to a pCWJ vector;

(2) transferring the recombinant vector into competent cells (whole-body gold biotechnology, Inc.) of Escherichia coli Trans 1T1, coating on LB plate with 50mg/L chloramphenicol resistance, and culturing at 37 deg.C overnight;

(3) and (3) selecting a single colony growing on the plate, transferring the single colony to an LB culture medium containing 50mg/L chloramphenicol resistance, extracting a plasmid, and performing enzyme digestion verification by using restriction enzymes Spe I and Kpn I to finally obtain the recombinant plasmid pCWJ-YjhG.

With reference to the above procedure, the recombinant plasmids PCWJ-yjhG-adhP and pTRC99a-xylB-mdLC were prepared successively.

Example 2 production of 1,2, 4-butanetriol by fermentation of recombinant E.coli Using xylose as substrate

Genetically engineered bacteria containing the recombinant plasmid pTRC99a-xylB-mdLC and the recombinant plasmid PCWJ-yjhG-adhP are streaked and activated on an LB plate (streptomyces resistance and ampicillin resistance), single colonies are picked up and inoculated into 5 mL of LB culture medium, and the culture is carried out at 37 ℃ and 200rpm for 7-10 h.

A group of 500ml triangular flasks are respectively filled with 50ml of fermentation medium containing 10g/L xylose, seed liquid prepared by recombinant escherichia coli is inoculated into the fermentation medium according to the inoculation amount of 4% volume ratio, IPTG is added when the culture is carried out at 37 ℃ till OD600=2, the final concentration is 1mmol/L, IPTG is added and then the culture is carried out at 33 ℃ till the fermentation is finished, samples are taken every 12 h, and the yield of D-1,2, 4-butanetriol is measured, and the result is shown in figure 1. As can be seen, the production rate is obviously reduced after the strain is fermented for 64 hours, and the yield is not increased any more.

The formula of the fermentation medium is as follows: 5g/L yeast powder, 10g/L peptone, 10g/L sodium chloride, 5g/L glucose and 10g/L xylose.

Example 3 production of 1,2, 4-butanetriol by recombinant Escherichia coli simultaneous saccharification and fermentation Using corncobs as substrates

Crushing corncobs, sieving the crushed corncobs with a 40-mesh sieve, mixing 1mol/L sodium hydroxide solution with the corncobs according to the solid-to-liquid ratio of 10%, carrying out high-temperature treatment on the mixed suspension hydrolysate, filtering to obtain corncob residues, and washing the residues to be neutral by using pure water.

Preparing LB culture medium of 5g/L yeast powder, 10g/L peptone and 5g/L sodium chloride. Subpackaging in 500mL conical flasks, filling liquid in 50mL, adding 2g of pretreated corncob residues into LB culture medium to prepare a fermentation culture medium, and sterilizing for later use.

Genetically engineered bacteria containing the recombinant plasmid pTRC99a-xylB-mdLC and the recombinant plasmid PCWJ-yjhG-adhP are streaked and activated on an LB plate (streptomyces resistance and ampicillin resistance), single colonies are picked up and inoculated into 5 mL of LB culture medium, and the culture is carried out at 37 ℃ and 200rpm for 7-10 h.

Taking a group of 500ml triangular flasks, respectively filling 50ml fermentation culture medium containing 2g of corncob residues, inoculating seed liquid prepared by recombinant escherichia coli into the fermentation culture medium according to the inoculation amount of 4% volume ratio, culturing at 37 ℃ for 2h, adding IPTG (isopropyl-beta-thiogalactoside) until the final concentration is 1mmol/L, culturing at 33 ℃ after adding IPTG until the fermentation is finished, taking samples every 12 h, and measuring the yield of D-1,2, 4-butanetriol. The results are shown in FIGS. 2 and 3. As shown in figure 2, the total amount of xylose produced by 2g of pretreated corncob residues after enzymolysis for 24h reaches 0.52g, the concentration is 10.45g/L, which is 8.75 times of that of xylose produced by direct enzymolysis of the corncobs, and the alkaline pretreatment effect is good. As shown in figure 3, the simultaneous saccharification and fermentation by using the corncob as the substrate not only improves the yield of the butanetriol, but also continuously produces the butanetriol after 64 hours, thereby proving that the corncob can continuously provide the substrate for the fermentation process.

The D-1,2, 4-butanetriol is produced by using the corncobs for simultaneous saccharification and fermentation, and the corncob enzymolysis and the fermentation process are coupled, so that the yield of the D-1,2, 4-butanetriol is improved, and a new process route is provided. The corn cob has extremely high annual output and low price in China, the corn cob is greatly used, the production cost is greatly saved, the operation process is simple, and the corn cob has the potential of large-scale production.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.